Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin

Abstract

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.